De Novo design of α-helical heme binding proteins capable of versatile cofactor ligation.

De novo design of artificial metalloproteins offers a powerful approach to dissect and mimic the diverse and exquisite coordination environments found in natural heme proteins. These designed heme proteins seek to replicate the broad array of metabolic, regulatory, and structural functions that hemes perform in biological systems. In this chapter, we present an amenable methodology for the preparation and characterization of de novo-designed heme-binding coiled coils featuring either His- and/or Cys-based axial ligation, which mimic the redox active sites of peroxidases, chloroperoxidases, and cytochrome P450 monooxygenases. The ability to reversibly control heme coordination and spin state through pH variations within a single scaffold provides novel insights into the principles governing catalysis in heme-containing and heme-binding proteins and paves the groundwork for engineering versatile catalysts with tailored reactivity.